Neural transplantation using pluripotent neuroepithelial cells

ABSTRACT

The subject invention pertains to a novel method of correction of behavioral and/or psychological deficits made possible by the intracerebral transplantation of pluripotent neuroepithelial cells. Cells, cell lines, pharmaceutical preparations, medicaments, methods for the production and maintenance of the cell lines for use in the method of the invention are encompassed by the invention.

[0001] The present application relates to the correction of behaviouraland/or psychological deficits by the intracerebral transplantation ofneural cells, and to cells and medicaments therefor. The invention alsoconcerns methods for the production and maintenance of the cell lines.

[0002] Behavioural and/or psychological deficits are caused by manydiseases and may also be caused when the brain undergoes trauma. Forexample, motor dysfunction is one symptom of Parkinson's disease. Asyet, in most cases, there is no satisfactory treatment available.

[0003] The present invention provides for a method of treatment of abehavioural and/or psychological deficit which comprises intracerebraltransplantation of a therapeutically effective amount of pluripotentneuroepithelial cells.

[0004] The present invention is based in part on the observation that,when transplanted into a damaged or diseased brain, pluripotentneuroepithelial cells appear to respond to signals from the damaged ordiseased brain by taking up a phenotype that is able to replace orcompensate for functional deficits to which the damage or diseaseotherwise leads.

[0005] The term “pluripotent” is used herein to denote a anundifferentiated neuroepithelial cell that has the potential todifferentiate into different types or different phenotypes of cell, inparticular into cells having the appropriate phenotype for the intendeduse. The cell type or phenotype into which such a pluripotent cellfinally differentiates is at least partly dependent on the conditions inwhich the cell exists or finds itself.

[0006] For use in the present invention the neuroepithelial cells shouldbe capable of differentiating into cells appropriate to repair orcompensate for damage or disease in the target area of the brain. Itwill be appreciated that cells for transplantation need not be capableof differentiation into all types or phenotypes of neural cells. Thecells may, for example, be bipotent. However, a high degree of potencyis generally preferred as this gives greater flexibility and potentialfor transplantation into different areas of the brain.

[0007] Suitable pluripotent cells include those called or known as “stemcells” and those called or known as “precursor cells”.

[0008] The pluripotent neuroepithelial cells are advantageously, andwill generally be, conditionally immortal.

[0009] The treatment may be carried out on any mammal but the presentinvention is especially concerned with the treatment of humans,especially treatment with human cells, and with human cells and celllines.

[0010] The present invention provides a mammal which has undergonetreatment according to the present invention.

[0011] The present invention provides isolated human, pluripotentneuroepithelial cells.

[0012] The present invention especially provides human, conditionallyimmortal pluripotent neuroepithelial cells.

[0013] The present invention further provides a conditionally immortal,pluripotent, neuroepithelial cell line, especially for therapeutic use,more especially for the treatment of a behavioural and/or psychologicaldeficit.

[0014] The cells of the present invention are capable of correcting abehavioural or psychological deficit when implanted into a damaged partof the human brain. The term “damage” used herein includes reduction orloss of function. Damage may be caused by any of a variety of meansincluding physical trauma, hypoxia (lack of oxygen), chemical agents,for example, damage may be caused by drug abuse, and disease. Thefollowing diseases and pathological conditions are examples of diseasesor conditions which result in behavioural and/or psychological deficitswhich may be treated in accordance with the present invention: traumaticbrain injury, stroke, perinatal ischaemia, including cerebral palsy,Alzheimer's, Pick's and related dementing neurodegenerative diseases,multi-infarct dementia, Parkinson's and Parkinson's type diseases,Huntington's disease, Korsakoff's disease and Creuzfeld-Jacob disease.Amnesia, particularly following transitory global ischaemia such asafter cardiac arrest or coronary bypass surgery, may also be treated inaccordance with the present invention.

[0015] The present invention further provides a process for theproduction of human, conditionally immortal, pluripotent,neuroepithelial cells which comprises the steps of:

[0016] (a) obtaining neuroepithelial cells from a human fetus, the cellsbeing at a stage early enough in the developmental pathway that theyhave the ability to differentiate into a variety of different brain celltypes,

[0017] (b) introducing into those cells DNA which comprises a sequencecapable of causing the cells to be conditionally immortal under thecontrol of appropriate control elements, and

[0018] (c) maintaining the cells in vitro under permissive conditions.

[0019] The process may further include the step of cloning the cells toobtain one or more cell lines.

[0020] A further aspect of the invention provides for pluripotent,neuroepithelial cells, optionally in isolated form, for therapeutic use,especially in humans. The therapeutic use may be treatment of abehavioural and/or psychological deficit.

[0021] A further aspect of the invention provides for conditionallyimmortal, pluripotent, neuroepithelial cells for therapeutic use,especially in humans. The therapeutic use may be treatment of abehavioural and/or psychological deficit.

[0022] The present invention further provides for the use ofpluripotent, neuroepithelial cells, optionally in isolated form, in themanufacture of a medicament for the treatment of a behavioural and/orpsychological deficit. The medicament to be administered comprisespluripotent neuroepithelial cells.

[0023] The present invention especially provides for the use ofconditionally immortal, pluripotent, neuroepithelial cells in themanufacture of a medicament for the treatment of a behavioural and/orpsychological deficit. The medicament to be administered comprisesconditionally immortal, pluripotent, neuroepithelial cells.

[0024] The conditionally immortal cells according to, and used in, thepresent invention may be from clonal cell lines or may be of mixedpopulation. Cells from clonal cell lines may be preferred. Cells from asingle cell line may be used or a mixture of cells from two or more celllines may be used.

[0025] The invention further provides a pharmaceutical preparationcomprising cells according to the invention and a pharmaceuticallyacceptable carrier.

[0026] Transplantation of conspecific fetal neural tissue into a damagedbrain has been studied in animal experiments and consequent repair hasbeen observed at the neuroanatomical, physiological and behaviourallevels (Dunnett & Bjorklund, 1994). There has been some application ofthis work in the treatment of the motor dysfunction of Parkinson'sdisease (Lindvall, 1994) but widespread use of this technique ishandicapped by the need for tissue derived from conspecific fetal brain.The fetal tissue required must be specific to the type of damage oneaims to repair and it must be taken at a precise, time-limited stageduring brain development that differs according to both brain region andcell type. This leads to both practical and ethical problems.

[0027] Work on fetal tissue transplant (Sinden, 1995) in certain typesof damage has shown that very specific matching of cell types isrequired to obtain improvement in cognitive function.

[0028] We have found that when conditionally immortal pluripotentneuroepithelial cells are implanted into a damaged brain the cellsdifferentiate into the correct form of cell required to repair thedamage and the differentiated cells are able to form the appropriateconnections required to improve function. The phenotype of thedifferentiated cells may be the same as the phenotype of the damaged orlost cells, however, the differentiated cells may be of a differentphenotype, or of a number of phenotypes. In any case, the cells take upa phenotype that is capable of functionally integrating and compensatingfor the damaged or lost cells. That is assisted by the propensity, thatwe have discovered, of the cells to migrate to, and seek out, damagedtissue.

[0029] The use of pluripotent cells means that with one clonal cell lineit is possible to repair damage in number of different areas of thebrain. It also means that if more than one particular cell type isrequired to repair damage in a given area then a single pluripotent cellline will be capable of differentiating into the different types ofcells required.

[0030] Conditionally immortal cells are cells which are immortal undercertain permissive conditions but are not immortal under nonpermissiveconditions. In the present case this means that by conditionallyimmortalising pluripotent precursor cells extracted from fetal tissueand maintaining them under permissive conditions the development of theprecursor cells may be arrested at a chosen stage and they may bepropagated for long periods. Use of conditionally immortalisation allowsthe development of clonal lines which are readily expandable in vitro.If the conditions under which the cells are maintained are switched tononpermissive conditions, the development of the cells is allowed tocontinue. If the correct conditions are provided the cells will continueto develop and will differentiate.

[0031] Immortalised cells are usually prepared by the transduction of anoncogene into cells. There is therefore a risk of tumour formation inthe long term, so such cells are not preferred for use in the presentinvention.

[0032] Conditionally immortal cells have the advantages of immortalcells in that they are “frozen” in the desired stage of development, areeasily maintained and multiply well when under permissive conditions butthey may be used in transplants as long as the environment into whichthey are transplanted has nonpermissive conditions. In the case of thecells of the present invention the gene used to confer conditionalimmortality should be chosen so that the conditions present in the brainwill correspond to nonpermissive conditions.

[0033] The usual way to immortalise the cells is by transduction of anoncogene. The use of conditionally immortal cells means that undernonpermissive conditions the cells do not have oncogenic properties andso this excludes any possibility of the implantation of cells leading totumour growth.

[0034] If non-immortal cells are used then these may be maintained invitro in culture media with the addition of growth factors.

[0035] The gene which is used to confer conditional immortality may beincorporated into cells after extraction from a fetal animal.Alternatively, transgenic animals, other than humans, whose neuralepithelial cells comprise a gene for conferring conditional immortalitymay be prepared and bred. If transgenic animals are bred then the cellscollected from the fetal animal tissue are already conditionallyimmortal and do not require further treatment.

[0036] The cells used in the treatment of humans should preferably bederived from human cells to reduce problems with immune rejection. Thisrequires the use of fetal tissue. The use of conditionally immortalcells means that once a population of cells has been established it isnot necessary to use fetal tissue again. For example, cells are takenfrom a human fetus at the appropriate stage of development and the DNAnecessary to cause conditional immortality in the cells is inserted.Those cells may then be propagated or they may be cloned and individualcell lines selected. Maintenance of the mixed populations and/or ofselected cell lines provides a constant source of material forimplantation.

[0037] To treat a patient it is generally of assistance to know wheredamage has occurred in the brain. Once the existence of damage has beenestablished, whether it be in one isolated area or in several areas,treatment by implantation of cells into the damaged area may be carriedout. In many cases, however, the location and/or type of damaged tissuemay be unknown or only poorly characterised. For example,neurodegenerative diseases may lead to widespread damage to differenttypes of cells. Treatment of such damage is still possible and isassisted by the ability of pluripotent neuroepithelial cells to migrateextensively once transplanted and to seek out damaged tissue. Thepluripotent cells may be transplanted at a single site, or preferably atmultiple sites, and are able to migrate to the site(s) of damage and,once there, differentiate in response to the local microenvironment,into the necessary phenotype or phenotypes to improve or restorefunction. Post mortem analysis of the brains of rats that had receivedtransplants of cells of a conditionally immortal pluripotentneuroepithelial cell line showed that the cells aggregated in the areaof the damaged tissue, see Example 9 below, thus illustrating thepropensity of the cells to establish and integrate themselves in thearea of damage rather than in an area of undamaged tissue. Thepluripotent nature of the cells and their propensity for damaged tissuemeans that treatment with cells from a single cell line of high potencyis able to lead to compensation for widespread damage of a number ofdifferent cell types.

[0038] After treatment the progress of the patient may be monitoredusing behavioural and/or psychological tests and/or, if desired, testswhich monitor brain activity in selected areas of the brain. Forexample, tests for cognitive function may be performed before and aftertransplantation.

[0039] Preferably, treatment will substantially correct a behaviouraland/or psychological deficit. However, that may not always be possible.Treatment according to the present invention and with the cells,medicaments and pharmaceutical preparations of the invention, may leadto improvement in function without complete correction. Such improvementwill be worthwhile and of value.

[0040] The number of cells to be used will vary depending on the natureand extent of the damaged tissue. Typically, the number of cells used intransplantation will be in the range of about one hundred thousand toseveral million. Treatment need not be restricted to a singletransplant. Additional transplants may be carried out to further improvefunction.

[0041] The present invention is illustrated by work we have carried outon rats which have brain damage. In the experiments described belowconditionally immortal cells used for transplantation are derived fromthe H-2 K^(b)-tsA58 transgenic mouse developed by M. Noble and hisassociates at the Ludwig Institute for Cancer Research (Jat et al.,1991). All cells from this mouse possess a temperature-sensitiveoncogene (tsA58, the temperature sensitive mutant of the SV40 large Tantigen under the control of the interferon-inducible H-2 K^(b)promotor) such that the cells divide at the permissive temperature(lower than body temperature, 33° C.) but differentiate only whenrestored to mouse body temperature (38° C.-39° C.). It is this featurethat provides them with conditional immortality. This allowed us toclone and expand cell lines in vitro which then differentiated upontransplantation into a host brain. A number of cell lines were clonedfrom a population of cells taken originally from the transgenic mouse,specifically, from embryonic day 14 (E 14) hippocampus. We studied therats, which received transplants of those cells, for at least 8 monthsand in no case did the cells, after transplantation, form tumours.Furthermore, in post-mortem histological preparations, the transplantedcells (marked by prior transfection with a lac-z reporter gene) have theappearance of differentiated cells appropriate to the rodent nervoussystem.

[0042] The cloned cell lines show the potential, in vitro, todifferentiate into more than one phenotype, e.g., into both astroglialand neuronal phenotypes, see Example 4 below.

[0043] The lesion-and-behaviour model in which we have demonstrated thatcloned cell lines are able to restore function in the damaged brain isone that we have previously studied intensively using fetal conspecifictransplants (see Sinden et al., 1995). It utilises rats in which thetechnique for four-vessel occlusion (4 VO), simulating human heartattack, causes relatively circumscribed and specific damage to the CA 1pyramidal cells of the dorsal hippocampus, along with a cognitivedeficit manifest as difficulty in locating a submerged and invisibleplatform in a swimming pool. This lesion and behaviour model provides amodel of cognitive dysfunction occurring as a consequence of a commonform of brain damage, i.e., transient loss of blood supply to the brain,for example, as may occur during cardiac arrest.

[0044] We have previously demonstrated that, for fetal cell-suspensiontransplants to restore performance in this task, they must be highlyspecific to the damage caused by 4VO: transplants containing CA 1pyramidal cells are effective; transplants containing cholinergic cellsfrom the basal forebrain, granule cells from the dentate gyrus, or evena different class of pyramidal cells (CA3) from the hippocampus areineffective. Examples 5 to 8 below described experiments in which boththe clonal cell lines and a mixed population of E 14 hippocampalneuroepithelial cells taken from the H-2 K^(b)-tsA58 transgenic mouseprovide effective transplants for restoration of cognitive function inthis model.

[0045] We have found that two of the three clonal cell lines tested, theMHP36 cell line (previously known as the C36 cell line) and the MHP3cell line are as effective at restoring cognitive function as fetal rattransplants containing CA 1 pyramidal cells. The third cell line tested,the MHP15 cell line (previously known as the C15 cell line) leads to animprovement of function but does not show as great an improvement asMHP36 and MHP3. The chances that we happened to pick upon cell linesthat would differentiate into CA 1 pyramidal cells, irrespective of thenature of the host brain environment, are small. Thus it appears thatthe cell lines are capable of responding to damage-associated signals soas to differentiate into cells, of one or more types, that are able tore-establish the necessary connections and restore the function(s)discharged by the damaged tissue. It is this capacity that provides botha strategy and a material basis for transplant therapies with which totarget a wide range of behavioural and psychological deficits consequentupon an equally wide range of forms of damage to the human brain, whilecircumventing the ethical and practical problems associated with the useof human fetal tissues.

[0046] The two cell lines which have shown the greatest ability, so far,to restore function are both FGF2-responsive, i.e., they substantiallyincrease their proliferation in both permissive and non-permissiveculture in the presence of that growth factor, whereas the third cellline is only slightly responsive. Cells and cell lines which showsignificantly increased proliferation in response to the addition of agrowth factor to their culture environment are therefore generallypreferred. The cells may be tested under permissive conditions and/ornonpermissive conditions. Cells showing the greatest increase inproliferation are generally most preferred. The growth factor used totest the cells should preferably be appropriate to the area of the brainin which the cells are intended for use, i.e., a growth factor secretedin that area. For example, cells intended for the repair of tissue inthe hippocampus may be tested with FGF2 (also known as bFGF). Cellsresponsive to FGF2 are generally preferred. Other mitogenic growthfactors may be used in testing, including EGF and NGF.

[0047] The invention therefore provides a method of testing comprisingmaintaining a population of cells of a conditionally immortalpluripotent neuroepithelial cell line in vitro and culturing portions ofthe cells under permissive conditions, in the presence and absence of agrowth factor, for example, FGF2, and determining the proliferation ofthe cells. Preferably the cells are also tested under nonpermissiveconditions. Those cells which are responsive, i.e., show significantlyincreased proliferation in the presence of the growth factor under bothpermissive conditions, and preferably also under nonpermissiveconditions, appear to be cells which are especially suitable for use inthe treatment of the invention. The growth factor may, for example, beused at a concentration of long/ml.

[0048] The temperature-sensitive oncogene which confers conditionalimmortality upon cells derived from the H-2 K^(b)-tsA58 transgenic mousecan be introduced into human cells in vitro. Well known techniques forthe introduction of exogeneous DNA exists and these may be used, forexample, the gene may be introduced by transfection of the cells. Normalscreening techniques for checking that the gene has been incorporatedmay be used, for example, Southern blotting may be used to screen forDNA insertion sites. In some cases markers may be used or, if the tsSV40 large T antigen gene is used then cells may be screened at thepermissive temperature for expression of SV40 as described in Example 4.

[0049] It should be understood that although the experiments describedin the Examples below have been carried out using the ts SV40 large Tantigen gene to confer conditional immortality on the cells, any othergene which is capable of causing conditional immortality may be used.Such genes may be constructed from known oncogenes. For example, aconditionally immortal gene has been constructed from the c-myc oncogeneand is described by Hoshimaiuaru et al, 1996.

[0050] In the experiments on rats which are described in Examples 6, 7and 8 below conditionally immortal pluripotent cells have been used torepair a very specific type of damage. The uses of cells according tothe invention are not limited to repair of that particular type ofdamage. Transplantation into any area of the brain is envisaged withconsequent improvement in function.

[0051] The part of the fetal brain from which the neuroepithelial cellsare taken and the precise time (stage and development) may vary. Ifpluripotent cells are desired then the cells must, however, be taken ata point early enough in the developmental pathway that they have theability to differentiate into the desired variety of different typesand/or phenotypes of brain cell types. For example, in the case of cellstaken from the embryonic mouse hippocampus the cells may be taken onembryonic day 14 to 15. Human cells may be taken at the equivalentdevelopmental stage. For example, cells may be taken from human fetusesat about 8 weeks.

[0052] Cells which have been removed may be screened in vitro to ensurethat they are still able to differentiate, in particular, todifferentiate into the appropriate type or phenotype of cell. Differentareas of the brain when damaged may produce different signals, forexample, growth factors, and/or different types of damage may causedifferent signals. The ability to differentiate may be determined invitro in the presence of the appropriate signal, for example, theappropriate growth factor. Example 4 below describes a procedure inwhich the ability of cells to differentiate into neuronal and glialphenotypes may be shown.

[0053] Some behavioural and/or psychological deficits are caused by theabsence of one or more chemicals in an otherwise healthy brain. It haspreviously been proposed that transplants of transgenic cells could beused to supply the missing chemicals. The present invention is notspecifically concerned with such problems. Although the cells of thepresent invention may be genetically modified to include extra geneswhich express desired products, this will not usually be necessarybecause the cells used according to the present invention, oncetransplanted, differentiate and then function fully as replacements forcells which have been lost or damaged. The cells achieve functionalintegration and replace or compensate for the missing or damaged cells.They become a functional part of the brain rather than being merely asophisticated method of drug administration. Genetic modification of thecells will therefore usually be restricted to the insertion of genesnecessary for conditional immortalisation and cloning. Genes requiredfor cloning may be, for example, a gene providing resistance to aselected antibiotic to enable selection. Genetic modification to enablesecretion of pharmacologic agents is not preferred.

[0054] Methods for transplantation of cells into humans and animals areknown to those in the art and are described in the literature in theart. The term “transplantation” used herein includes the transplantationof cells which have been grown in vitro, and may have been geneticallymodified, as well as the transplantation of material extracted fromanother organism. Cells may be transplanted by implantation by means ofmicrosyringe infusion of a known quantity of cells in the target areawhere they would normally disperse around the injection site. They mayalso be implanted into ventricular spaces in the brain. If implantedinto the neonate then they may disperse throughout the entire brain.

[0055] The phrase “intracerebral transplantation” used herein includestransplantation into any portion of the brain. Transplantation is notrestricted to the front and larger part of the brain.

[0056] The following non-limiting Examples illustrate the invention.

[0057] FIGS. 1 to 20 show the results of the experiments described inExamples 3 and 5 to 8. The figures are as follows:

[0058]FIG. 1—shows the proliferation of MHP15 cells at 33° C. and 39° C.in SFM.

[0059]FIG. 2—shows the proliferation of MHP15 cells at 33° C. and 39° C.in SFM with gamma-interferon (12U/ml).

[0060]FIG. 3—shows the proliferation of MHP15 cells at 33° C. and 39° C.in SFM with FGF2 (10 ng/ml).

[0061]FIG. 4—shows the proliferation of MHP15 cells at 33° C. and 39° C.in SFM with gamma-interferon (12U/ml) and FGF2 (10 ng/ml).

[0062]FIG. 5—shows the proliferation of MHP36 cells at 33° C. and 39° C.in SFM.

[0063]FIG. 6—shows the proliferation of MHP36 cells at 33° C. and 39° C.in SFM with gamma-interferon (12U/ml).

[0064]FIG. 7—shows the proliferation of MHP36 cells at 33° C. and 39° C.in SFM with FGF2 (10 ng/ml).

[0065]FIG. 8—shows the proliferation of MHP36 cells at 33° C. and 39° C.in SFM with gamma-interferon (12U/ml) and FGF2 (10 ng/ml).

[0066]FIG. 9—shows the latency of rats in a Morris water maze over timefor sham-lesioned control rats which received sham grafts (CON),ischaemic rats which received sham grafts (ISC), ischaemic rats whichreceived an implant of CA1 cells (CA1), CA3 cells (CA3), or of a mixedpopulation of conditionally immortal hippocampus precursor cells takenfrom a H-2 K^(b)-tsA58 transgenic mouse (tsA58).

[0067]FIG. 10—shows the latency of rats in a Morris water maze over timefor sham-lesioned control rats which received sham grafts (CON),ischaemic rats which received sham grafts, (ISC), ischaemic rats whichreceived an implant of CA1 cells (CA1), of cells of the MHP36 cell line(MHP36) or of a mixed population of conditionally immortal hippocampusprecursor cells taken from a H-2 K^(b)-tsA58 transgenic mouse (tsA58).

[0068]FIG. 11—shows the latency of rats in a Morris water maze over timefor sham-lesioned control rats which received sham grafts (CON),ischaemic rats which received sham grafts (ISC) and ischaemic rats whichreceived an implant of cells of the MHP36 cell line (passage 24 to 32)(MHP36).

[0069]FIG. 12—shows the latency of rats in a Morris water maze over timefor sham-lesioned control rats which received sham grafts (CON),ischaemic rats which received sham grafts (ISC), ischaemic rats whichreceived an implant of cells of the MHP36 cell line (MHP36), of cells ofthe MHP15 cell line (MHP15) or of cells of the MHP3 cell line (MHP3).

[0070]FIG. 13—shows the proliferation of MHP15 cells at 33° C. and 39°C. in SFM, as in FIG. 1 but for days 0 to 6.

[0071]FIG. 14—shows the proliferation of MHP15 cells at 33° C. and 39°C. in SFM with gamma-interferon (12U/ml), as in FIG. 2 but for days 0 to6.

[0072]FIG. 15—shows the proliferation of MHP15 cells at 33° C. and 39°C. in SFM with FGF2 (10 ng/ml), as in FIG. 3 but for days 0 to 6.

[0073]FIG. 16—shows the proliferation of MHP15 cells at 33° C. and 39°C. in SFM with gamma-interferon (12U/ml) and FGF2 (10 ng/ml), as in FIG.4 but for days 0 to 6.

[0074]FIG. 17—shows the proliferation of MHP36 cells at 33° C. and 39°C. in SFM, as in FIG. 5 but for days 0 to 6.

[0075]FIG. 18—shows the proliferation of MHP36 cells at 33° C. and 39°C. in SFM with gamma-interferon (12U/ml), as in FIG. 6 but for days 0 to6.

[0076]FIG. 19—shows the proliferation of MHP36 cells at 33° C. and 39°C. in SFM with FGF2 (10 ng/ml), as in FIG. 7 but for days 0 to 6.

[0077]FIG. 20—shows the proliferation of MHP36 cells at 33° C. and 39°C. in SFM with gamma-interferon (12U/ml) and FGF2 (10 ng/ml), as in FIG.8 but for days 0 to 6.

EXAMPLE 1

[0078] Preparation of Mixed Population Cultures

[0079] Hippocampi were dissected from E14H-2 K^(b)-tsA58 mice. A cellsuspension was prepared after both trypsinisation and mechanicaldissociation and cells were plated at a concentration of 45×10³ to50×10³ cells/ml into 10 cm² culture dishes. The cells were cultured inDMEM: F12 serum-free medium (SFM) with the following additions: bovineserum albumen (0.0286%); transferrin (0.1 mg.ml); putrescine (16.2μg/ml); insulin (5 μg/ml); progesterone (0.062 μg/ml); selenium (0.0383μg/ml); L-glutamine (2 mM); L-thyroxine (0.4 μg/ml); tri-iodothyronine(0.337 μg/ml); heparin (lOUSP units/ml); penicillin/streptomycin(10,000:1000 units/ml) all obtained from Sigma. In addition basicfibroblast growth factor, previously known as bFGF and now known asFGF2, (FGF2-10 ng/ml) and gamma-interferon (g-IFN-12U/ml) were added tothe cells and they were incubated at 33° C. in 5% CO₂. Every 2 to 3 dayshalf the medium was replaced and all the FGF2 and g-IFN.

EXAMPLE 2

[0080] Production of Clonal Lines

[0081] pPGKB-geo plasmid (obtained from P. Soriano) which consisted of alacZ and neomycin resistance fusion driven by a pGK promoter was grownovernight at 37° C. in Luria Bertani broth and then purified using acommercially available kit (Qiagen, Germany). The purified plasmid DNAwas linearised using the restriction enzyme Sal 1 (Promega, U.K.) thensterilised and resuspended in the TE buffer at a final concentration of1 mg/ml. A mixed population of hippocampal neuroepithelial cells asprepared in Example 1 were electroporated in order for the cells toincorporate the lacZ fusion gene.

[0082] Cells were seeded at a concentration of 10⁵ to 10⁶ cells/plate inSFM (with the additions listed in Example 1) with the appropriate FGF2and g-IFN additions and after 24 hours the medium was replaced with SFMcontaining G418 (a neomycin-like antibiotic) (200 μg/ml) which wouldallow only those cells which had incorporated the plasmid correctly tobe able to express G418 resistance. Medium was replaced 2 to 3 times aweek and the cells were left for 4 to 6 weeks to allow clones todevelop.

[0083] Clones that were selected were discrete from one another andpicked using glass cloning rings which had been dipped in silicongrease. The clonal cells were transferred to 24 well plates after abrief incubation of 5 mins with EDTA/EGTA (1:100) solution at 37° C.After several days these cells became confluent and were transferredinto 6 well plates and then into 10 cm² culture dishes, thereafter beingtreated exactly the same as the mixed population non-transfected lines,as described above, with the exception of the G418 additions. Of the 32clones picked, 9 clones were expanded into permanent cell lines.

[0084] All of these clonal hippocampal lines were shown to beconditionally immortal; cell counts by chromogenic MTT assay showed1-2×10³-fold proliferation from original plating densities in permissiveconditions in serum-free media without added FGF2 at 2 to 6 days invitro (hereinafter referred to as “DIV”) with a rapid reduction inplated cell numbers at nonpermissive conditions (39° C., gamma-IFN). Thehippocampal neuroepithelial population from which these lines werederived, however, required supplementation with FGF2 for proliferation.Two of the nine lines were, in addition, FGF2-responsive, substantiallyincreasing their proliferation rate in both permissive and nonpermissiveculture in the presence of this growth factor.

[0085] Lines have been maintained for multiple passages and frozenstocks have been thawed and cultured without change of phenotype.

EXAMPLE 3

[0086] Proliferation of Clonal Lines MHP15 and MHP36 in vitro

[0087] (Cell lines MHP36 and MHP15 were previously known as C36 and C15respectively. The C36 and C15 references were used in the applicationfrom which this current application claims priority. The cell lines arethe same cell lines, only the names have changed.) Cells from two of thepermanent cell lines were plated at a density of 8-12×10³ cells/wellinto 96 well plates in serum free DMEM:F12 medium (SFM) with theaddition of basic fibroblast growth factor (FGF2-10 ng/ml) andg-interferon (12U/ml) for 24 hours at 33° C. with 5% CO₂. Following thisperiod all of the medium, FGF2 and g-interferon was removed and eachcolumn consisting of 8 wells was treated with either:

[0088] (i) SFM with no supplements;

[0089] (ii) SFM with g-interferon (12U/ml);

[0090] (iii) SFM with FGF2 (10 ng/ml); or

[0091] (iv) SFM with both g-interferon (12U/ml) and FGF2 (10 ng/ml); for2, 4, 6, 8, and 14 days in vitro at either 33° C. or 39° C. Cells werecounted at each time point under each temperature and supplementcondition using a chromogenic MTT assay. Both cell lines show cleartemperature sensitivity in SFM both with and without g-interferon; cellsrapidly proliferate up to 200 fold plating density at the permissivetemperature (33° C.), but show minimal proliferation at thenon-permissive temperature (39° C.). The addition of FGF2 enhancesproliferation at both temperatures in the MHP36 line; but has only aslight effect on the MHP15 line; indicating the cell lines differ intheir responsiveness to this growth factor.

[0092] FIGS. 1 to 4 show the results for the MHP15 cell line for up to14 days and FIGS. 13 to 16 show the results in greater detail for days 0to 6. FIGS. 5 to 8 show the results for the MHP36 cell line for up to 14days and FIGS. 17 to 20 show the results in greater detail for days 0 to6.

EXAMPLE 4

[0093] MHP15 and MHP36 Clonal Cell Lines have Neuronal and GlialPotential when Differentiated in vitro.

[0094] Cells were prepared for immunocytochemistry using a range ofmarkers under both permissive conditions (33° C. plus g-interferon withadded FGF2) and nonpermissive conditions (39° C.) in SFM with theaddition of the differentiating agent dibutyryl cAMP (1 mM). 50-100×10³cells from each line were plated on fibronectin treated chamber slidesin SFM with FGF2 and g-interferon at 33° C. for 48 hours. Half theslides were fixed at this stage and the other half had media removed andsubstituted with SFM containing 1 mM dibutyryl cAMP and maintained at39° C. These slides were fixed after 2 to 8 days in vitro with 4%paraformaldehyde. The Table shows the proportion of all cells in 5randomly selected fields expressing the marker for progenitor cells,Nestin; the neuronal marker, neurone specific enolase (NSE); the glialmarker, glial fibrillary acidic protein (GFAP) and the marker for theimmortalising gene antigen, SV40. Both cell lines show neuronal andglial phenotypes after, but not before, differentiation. MHP15 CLONALLINE NESTIN NSE GFAP SV40 DAYS IN VITRO- % cells % cells % cells % cellsTEMPERATURE labelled labelled labelled labelled 2-33° C. 100 ± 0   0 ± 0  0 ± 0 100 ± 0 2-39° C. 100 ± 0 12.4 ± 2.2 3.8 ± 1 100 ± 0*

[0095] MHP36 CLONAL LINE NESTIN NSE GFAP SV40 DAYS IN VITRO % cells %cells % cells % cells TEMPERATURE labelled labelled labelled labelled2-33° C. 100 ± 0   0 ± 0   0 ± 0  100 ± 0 2-39° C. 100 ± 0 59.3 ± 7.012.2 ± 3.3 38.6 ± 16.1

[0096] Further Characterisation of MHP36 Clonal Line

[0097] The MHP36 cell line was further characterised at 2 DIV in bothpermissive and nonpermissive culture. It was found that the cellswere >95% X-gal labelled (i.e. showed a histochemical reaction to theβ-gal transduce marker) in permissive conditions. The cells were alsotested with two further antibodies, one for the neuronal marker PGP 9.5(Wilson et. al., 1988) and one for bromodeoxyuridine (BrdU) after onehour incubation with BrdU labelling medium as a marker for dividingcells. In permissive culture BrdU stained cells were found throughoutthe culture. There were no PGP 9.5 cells in permissive culture.

[0098] Following a switch to nonpermissive conditions in serum-freemedia (SFM) without additions, this cell line stopped dividing and themajority of cells died without differentiating into mature neuronal orglial phenotypes. However, in the presence of forskolin or retinoic acid(both of which are differentiating agents) the cultures could bemaintained for longer periods (5-14 DIV) and a proportion of the cellsshowed neuronal or glial phenotypes by 2 DIV. The MHP36 cells showed aflat-cell morphology in the presence of retinoic acid (10⁻⁹ M) with anumber of GFAP-positive fibrillary cells, SV40 expression was reduced to30% and BrdU staining to 5% of cells; no PGP 9.5 stained cells werefound. In the presence of forskolin (10⁻⁸ M), however, bipolar PGP9.5-positive cells were frequently found, many cells having longneuritic processes. GFAP-stained fibrils were not found in the cultures,SV40 expression was down-regulated (to 42% of total cells) and BrdUstaining was found in 4% of the culture. These findings further confirmthat MHP36 is a pluripotent neural precursor cell line whose lineagefate is at least partly determined by inductive signalling.

EXAMPLE5

[0099] Grafted H-2 K^(b-tsA)58 hippocampal precursor cells, like cellsuspensions of CAl cells, restore spatial learning and memory in ratswith ischaemic lesions of CA1.

[0100] The effects of grafts of cell suspensions of E19 CA1 (CA1), E19CA3 (CA3) and expanded cultures of E14 transgenic H-2 K^(b)-tsA58hippocampal neuroepithelial cells (tsA58) (harvested at Passage 5) onacquisition of a hidden platform position in the Morris water mazefollowing 15 minutes of 4VO ischaemia (ISC), producing selective lesionsto CA1 were studied. The methods described in the paper by Sinden et al,1995, especially those described in section 9 of the paper, werefollowed except where indicated to the contrary. The paper gives detailsof the method used to cause ischaemia, of methods of transplantation andof using the Morris water maze test. A brief summary of the procedure isgiven below.

[0101] Transient global ischemia was induced in male Wistar rats by the4VO method in which the vertebral arteries were electrocoagulatedthrough the alar formanae on the first cervical vertebrae, under 2%halothane anaesthesia (in 70% nitrous oxide and 30% oxygen), andsilastic ties were inserted around the carotids and brought to thesurface. Twenty four hours later the ties were tightened and clamped for15 minutes. Rats that failed to lose righting reflex within two minuteswere not included in the experiments. Wounds were rapidly sealed withclips and lignocaine applied.

[0102] H-2 K^(b)-tsA58 cells were removed from permissive culture andresuspended in Hank's balanced salt solution with 1 mMn-acetyl-L-cysteine ready for grafting. The cells were pulsed with 0.5μCi/ml ³H-Thymidine 48 hours before removal.

[0103] Grafts were bilaterally targeted to the dorsal CA1 cell field (2sites/hemisphere, 2 μl/site, 25K cells/μl for each cell-type graft) andrats were grafted 2 to 3 weeks after ischaemia surgery. The rats withgrafts of tsA58 cells were treated every other day for 14 dayspost-transplantation with the immunosuppressant, cyclosporin A (Sandoz,2.5 mg/rat in Cremophore EL, i.m.). Training (2 trials/day) began 12weeks post-transplantation (Number of subjects (Ns)=7 to 11/group).

[0104] Sham-lesioned control rats underwent cauterisation of thevertebral arteries but no ligation of the carotids and then receivedsham transplants. Sham transplants (grafts) were carried out by loweringthe graft injection needle to the appropriate site without making aninjection.

[0105] The water maze consisted of a black polypropylene circular pool(2 m diameter, 0.5 m high, with 0.25 m depth of water at 26° C.,rendered opaque with the addition of 200 ml milk). The escape platformwas a 9 cm clear perspex closed cylinder, located 0.02 m below the watersurface in the middle of the North West quadrant of the pool. At thestart of the trials, rats were placed in the pool facing the wall andallowed to swim until they found the platform, where they remained for10 seconds before removal. A rat which failed to find a platform within60 seconds was guided to it by the experimenter and the maximum latencyscored. Start positions were designated as North, South, East or Westand, in pseudorandom order, one start point close to the platform andone point distant from it were used each day. The swim path was recordedby an image analysing system (HVS Image, VP112), and digitally convertedinto a range of navigational measures.

[0106] The error bar indicates 2 standard errors between groups from theGroups X Days interaction of the analysis of variance. Latency to findthe platform for the ischaemic rats with both CA1 and tsA58 grafts wasnot significantly different from unlesioned controls (sham-lesionedcontrol rats which received sham grafts) (CON), whereas the otherischaemia groups with CA3 or sham grafts were significantly impairedrelative to the control, CA1 and tsA58 groups in latency and othermeasures of spatial learning. Post-mortem analysis showed similarselective CA1 neuronal loss (70-75%) from host CA1 in all ischaemicgroups including each of the grafted groups. Graft survival wasexcellent in all grafted groups.

[0107] Latency in the Morris water maze test is the time taken for asubject to swim to a hidden platform.

[0108] The results are shown in FIG. 9.

EXAMPLE 6

[0109] A clonal cell line (MHP36) derived from H-2 K^(b)-tsA58hippocampal precursor cells, restores spatial learning and memory inrats with ischaemic lesions of CA1.

[0110] The effects of grafts of cell suspensions of E19 CA1 (CA1),expanded cultures of E14 transgenic H-2 K^(b)-tsA58 hippocampalneuroepithelial cells (tsA58) (harvested at Passage 5) and an expandedclonal cell line (MHP36) derived from E14 immortal hippocampal precursorpopulation on acquisition of a hidden platform position in the Morriswater maze following 15 minutes of 4VO ischaemia (ISC), producinglesions of CA1 were studied. Methods were as described in Example 5.MHP36 cells were removed from permissive culture and resuspended inHank's balanced salt solution with 1 mM n-acetyl-L-cysteine ready forgrafting.

[0111] As in Example 5, grafts were bilaterally targeted to the dorsalCA1 cell field (2 sites/hemisphere, 2 μl/site, 25K cells/μl for eachcell-type graft) and graft surgery was conducted 7 to 14 days afterischaemia. All rats in this experiment (including ungrafted (sham-graft)and unlesioned (sham-lesioned) controls) were treated every other dayfor 14 days post-transplantation with the immunosuppressant, cyclosporinA (Sandoz, 2.5 mg/rat in Cremophore EL, i.m.). Training (2 trials/day)began 12 week post-transplantation (Ns=7-11/group). The error barindicates 2 standard errors between groups from the Groups X Daysinteraction of the analysis of variance. Replicating our firstexperiment, latency to find the platform for the ischaemic rats withboth CA1 and tsA58 grafts was not significantly different fromsham-lesioned controls, which received sham grafts, (CON). Moreover, theMHP36 clonal cell line grafted group was also not significantly impairedcompared to the control, CA1 and tsA58 groups. The results of theseexperiments are show in FIG. 10.

[0112] The experiments demonstrate that the transplanted precursor cellsappear to respond to signals from the damaged brain by taking up aphenotype able to replace or compensate for the functional deficits towhich the damage otherwise leads.

[0113] Further, analysis of variance (ANOVA) with repeated measuresshowed significant main effects of Groups (F_(4.38)=2.92, P<0.05),Blocks (F_(5.896)=36.50, P<0.001) and a significant Groups X Blockslinear coefficient interaction (F_(4.896)=3.23, P<0.02). t-Testcomparisons between linear coefficients revealed that the ischaemic-shamtransplant group had significantly impaired escape latency performancecompared to the control and the three grafted groups (minimum t₃₈=2.29,P<0.05); the control and the three grafted groups did not significantlydiffer among themselves (all t₃₈<1). ANOVAs of swim distances revealedsimilar outcomes to the latency results.

EXAMPLE 7

[0114] The water maze tests of Examples 5 and 6 were repeated in afurther set of experiments, see below. (In these experiments all ratswere immunosupressed by intramuscular injection of cyclosporin-A (2.5mg/rat in Cremophore EL) every other day for 15 dayspost-transplantation. The results are shown in FIGS. 11. In that figurethe mean time taken to find the platform is expressed as a function oftwo-day (4 trial) blocks of training. Bars-show 2×s.e.m. from the GroupX Blocks interaction of the analyses of variance.

[0115] These results further confirm that grafts of MHP36 cells are ableto reverse ischaemia induced learning deficits. Both control(sham-lesioned rats which received sham grafts) and MHP36 groups showedsignificantly faster escape latencies and shorter swim distances to theescape platform than the group with ischaemia which received shamgrafts.

[0116] An ischaemic group with grafts of MHP36 cells (P24-32) (N=12)(closed squares) was compared to ischaemic which received shamtransplants (N=10) (closed circles) and sham-lesioned controls whichreceived sham transplants (N=10) (open circles) on an identical watermaze procedure. The ANOVA of escape latencies showed significant maineffects of Groups (F_(2.29)=27.80, P<0.001), Blocks (F_(5.674)=54.72,P<0.001) and a significant Groups X Blocks interaction (F_(10.674)=5.81,P<0.001), with a significant linear coefficient interaction(F_(2.674)=23.31, P<0.001). t-Test comparisons between linearcoefficients revealed that the ischaemic-sham transplant group hadsignificantly impaired escape latency performance compared to both thecontrol and the MHP36 grafted groups (minimum t₂₉=5.54, P<0.001); thecontrol and the MHP36 group were not significantly different (t₂₉=1.01).ANOVAs on swim distance and other measures of spatial learning in thewater maze were entirely consistent with the escape latency results.

EXAMPLE 8

[0117] The experiments in Example 7 were repeated using grafts of cellfrom the MHP3 and MHP15 cell lines. MHP3 is one of the nine clonal celllines mentioned in Example 2 above and is one of the cell lines whichshowed responsiveness to FGF2. The results are shown in FIG. 12. Alsoshown in that figure are results for control rats (again sham-lesionedrats which received sham grafts), ischaemic rats which received shamgrafts and ischaemic rats which have received an implant of cells of theMHP36 cell line, as in Example 7 above. For MHP3 and MHP15 N=9. As maybe seen, grafts of MHP3 cells produce as effective learning performanceas those of MHP36, i.e., significantly faster than for the ischaemicgroup, but not significantly slower than for the control group. Graftsof MHP15 cells produce intermediate effects, i.e., significantly fasterthan for the ischaemic group, but also significantly slower than for thecontrol group.

EXAMPLE 9

[0118] Post-mortem ischaemic brain damage in Experiment 7A was assessedfrom cresyl violet stained sections by two independent observers incortex and striatum at two levels and in areas CA1-4 of the hippocampusat two further levels. Using a five-point scale, no damage was found inany region other than CA1, with the exception of two rats that showedmild CA3 cell loss. Independently of areas of grafted cells, average CA1cell loss at the level of maximal ischaemic damage (inter-aural anterior5.7 mm) ranged from 80% in the ischaemia plus sham transplant group to90% in the ischaemia plus CA1 graft group, with no significantdifferences among the ischaemia alone (sham-graft) andischaemia-plus-graft groups. Transplants of E19 fetal CA1 cells formed acircumscribed graft mass located above the CA1-damaged area, similar tothose previously reported. ³H-thymidine-labelled expanded populationcells were found dispersed throughout the entire hippocampal formation,the corpus callosum and the overlying neocortex; some clusters oflabelled cells were found within the lesioned CAl cell layer. Grafts ofX-gal-positive MHP36 cells had a much more restricted distribution:other than adjacent to the needle track, labelled cells were shown to beconfined to the hippocampus. This probably reflects both a greatersensitivity of the radioactive label, as well as a down-regulation ofB-gal expression over the long survival time in these experiments. Aspilot experiments had shown when grafts of MHP36 cells were made intounlesioned hippocampus, the X-gal-positive cells appeared to integrateinto all CA (but not dentate gyrus) neuronal cell-body layers. (Discretelabelled cells were found in the hippocampal tissue and particularly inareas CA3 and 4.) However, unlike the case of grafts into unlesionedhippocampus, dense aggregations of X-gal-stained cells were additionallyfound within the ischaemic CA1 cell layer. The degree of engraftmentvaried between rats such that aggregations were in a small proportion ofthe CA1 field, or almost fully populated the lesioned field within aparticular section, apparent on both X-gal- and nissl-stained sections.For example, some aggregations were found in the same hemisphere atdifferent points along the rostro-caudal axis of the damaged CA1 layer.From this it is clear that cells of the MHP36 cell line have apropensity to aggregate in the lesioned CA1 neuronal layer.

[0119] A time-course study of the migration of labelled grafted MHP36cells in ischaemic rats, using anti-B-gal immunohistochemistry toidentify grafted cells has shown that migration to and aggregation atCA1 is complete by 4 weeks post-grafting.

REFERENCES

[0120] Dunnett & Bjorklund, 1994, Functional Neural Transplantation,Raven Press, New York

[0121] Jat P. S. et al., 1991, P.N.A.S. (USA) 88, p5096

[0122] Lindvall, O, 1994, in Dunnett & Bjorklund, op. cit.

[0123] Sinden J. D. et al. (1995) Beh. Brain Sci. 18, p10

[0124] Wilson, P. O. G., Barber, P. C., Hamid Q., A. Power, B. F.,Dhillon A. P., Rode, J., Day, I. N. M., Thompson, R. J., and Polak J.M., Br. J. Exp. Pathol 69, p91-104 (1988).

[0125] Hoshimaiuaru, M., Ray, J., Sah, D. W. Y., Gage, F. H., PNAS USA,Vol.93, pl518-1523, (1996)

[0126] The present invention is not to be limited in scope by theExamples given above which are intended to illustrate the invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

[0127] The content of the above-mentioned literature references arehereby incorporated by reference.

We claim:
 1. A method of treating a behavioral or psychological deficitin an animal which comprises intracerebral transplantation of atherapeutically effective amount of pluripotent neuroepithelial cells tosaid animal.
 2. The method of claim 1, wherein tests for cognitivefunction are carried out before and after transplantation of saidpluripotent neuroepithelial cells.
 3. The method of claim 1, whereinsaid cells are conditionally immortal.
 4. The method of claim 1, whereinsaid cells are isolated.
 5. The method of claim 1, wherein said animalis a human.
 6. The method of claim 1, wherein said cells are from asingle cell line.
 7. The method of claim 1, wherein said cells are amixture of cells from two or more cell lines.
 8. The method of claim 1,wherein said cells have a high degree of potency.
 9. The method of claim1, wherein the proliferation of said cells is increased by the additionof FGF2 in vitro under both permissive and non-permissive conditions.10. The method of claim 1, wherein said cells differ from those found innature only in that said cells comprise exogenous DNA necessary toprovide conditional immortality, and optionally to allow cloning. 11.The method of claim 1, wherein said behavioral or psychological deficitis the result of hypoxia.
 12. The method of claim 1, wherein said cellsare human cells.
 13. Pluripotent, neuroepithelial cells for therapeutictreatment of an animal.
 14. The cells of claim 13, wherein said cellsare for therapeutic treatment of a behavioral or psychological deficitof said animal.
 15. The cells of claim 13, wherein said cells areconditionally immortal.
 16. The cells of claim 13, wherein said cellsare isolated.
 17. The cells of claim 13, wherein said animal is a human.18. The cells of claim 13, wherein said cells are from a single cellline.
 19. The cells of claim 13, wherein said cells are a mixture ofcells from two or more cell lines.
 20. The cells of claim 11, whereinsaid cells have a high degree of potency.
 21. The cells of claim 13,wherein the proliferation of said cells is increased by the addition ofFGF2 in vitro under both permissive and non-permissive conditions. 22.The cells of claim 13, wherein said cells differ from those found innature only in that said cells comprise exogenous DNA necessary toprovide conditional immortality, and optionally to allow cloning. 23.The cells of claim 14, wherein said behavioral or psychological deficitis the result of hypoxia.
 24. The cells of claim 13, wherein said cellsare human cells.
 25. A conditionally immortal, pluripotent,neuroepithelial cell line for therapeutic treatment of an animal. 26.The cell line of claim 25, wherein said cell line is for the treatmentof a behavioral or psychological deficit of said animal.
 27. The cellline of claim 25, wherein said animal is a human.
 28. The cell line ofclaim 25, wherein said cell line is from a single cell line.
 29. Thecell line of claim 25, wherein said cell line is a mixture of cells fromtwo or more cell lines.
 30. The cell line of claim 25, wherein cells ofsaid cell line have a high degree of potency.
 31. The cell line of claim25, wherein the proliferation of said cell line is increased by theaddition of FGF2 in vitro under both permissive and non-permissiveconditions.
 32. The cell line of claim 25, wherein said cell linediffers from cells found in nature only in that cells of said cell linecomprise exogenous DNA necessary to provide conditional immortality, andoptionally to allow cloning.
 33. The cell line of claim 26, wherein saidbehavioral or psychological deficit is the result of a transient loss ofblood supply to the brain of said animal.
 34. The cell line of claim 25,wherein cells of said cell line are human cells.
 35. A process for theproduction of human, conditionally immortal, pluripotent,neuroepithelial cells which comprises the steps of: (a) obtainingneuroepithelial cells from a human fetus, said neuroepithelial cellsbeing at a stage early enough in the developmental pathway that saidneuroepithelial cells have the ability to differentiate into a varietyof different brain cell types; (b) introducing into said neuroepithlialcells DNA which comprises a sequence capable of causing saidneuroepithlial cells to be conditionally immortal under the control ofappropriate control elements; and (c) maintaining said neuroepithelialcells in vitro under permissive conditions.
 36. The process of claim 35,which further includes the step of cloning said neuroepithelial cells toobtain one or more cell lines.
 37. A pharmaceutical compositioncomprising cells of claim 13 and a pharmaceutically acceptable carrier.38. A pharmaceutical composition comprising cells from the cell line ofclaim 25 and a pharmaceutically acceptable carrier.
 39. A pharmaceuticalcomposition comprising cells obtained according to the process of claim64 and a pharmaceutically acceptable carrier.
 40. A method of testingcomprising maintaining a population of cells of a conditionally immortalpluripotent neuroepithelial cell line in vitro and culturing portions ofsaid cells under permissive conditions in the presence and absence of agrowth factor and determining the proliferation of the cells.
 41. Themethod of testing according to claim 40, which further comprisesculturing portions of said cells under non-permissive conditions in thepresence and absence of a growth factor and determining theproliferation of said cells.
 42. A mammal which has undergone the methodof treatment according to claim
 1. 43. A cell line comprisingconditionally immortal, pluripotent, neuroepithelial stem cells, whereinsaid cell line is obtainable by culturing said stem cells underpermissive conditions in serum-free medium.
 44. The cell line of claim43, wherein said serum-free medium comprises a growth factor.
 45. Thecell line of claim 44, wherein said growth factor is FGF2.
 46. Cellsobtainable from a cell line of claim
 43. 47. The cells according toclaim 46, wherein said cells are for use in a method of therapeutictreatment of an animal.
 48. The cells according to claim 47, whereinsaid therapeutic treatment is a treatment of a behavioral orpsychological deficit of said animal.
 49. A method of treating an animalhaving a damaged brain, said method comprising intracerebraltransplantation of a therapeutically effective amount of a cell lineinto the damaged brain of said animal, said cell line comprisingconditionally immortal, pluripotent, neuroepithelial stem cells, whereinsaid cell line is obtainable by culturing said stem cells underpermissive conditions in serum-free medium into the damaged brain ofsaid animal.
 50. The method of claim 49, wherein said serum-free mediumcomprises a growth factor.
 51. The method of claim 49, wherein saidgrowth factor is FGF2.
 52. A method for treating a behavioral orpsychological deficit caused by damage to, or loss of, brain cells in amammal which comprises intracerebral transplantation to said mammal ofundifferentiated pluripotent cells having neuronal and glial potential,wherein said transplanted cells migrate and differentiate to replace, orcompensate for, said lost or damaged brain cells.
 53. The method ofclaim 52, wherein said undifferentiated pluripotent cells areconditionally immortal.
 54. The method of claim 52, wherein saidundifferentiated pluripotent cells are nestin-positive prior to saidintracerebral transplantation.
 55. The method of claim 52, wherein saidundifferentiated pluripotent cells are from a clonal cell line.
 56. Themethod of claim 52, wherein said behavioral or psychological deficit isthe result of hypoxia.